The invention relates to a flow cytometer which classifies leukocytes by irradiating a blood sample, as well as to a method for determining a detection angle range of the flow cytometer.
Leukocyte cells comprise various blood cells, such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Each of the cells consists of a nucleus and cytoplasm. The cells are known to differ in size and shape from each other, and particles included in cytoplasm, such as granules, are also known to differ in size, shape, and quantity from each other, depending on the type of cell. Granules or particles included in each cell have shown the following:    Lymphocyte: Very few granules of lymphocyte are present.    Monocyte: A considerably large number of granules, each having a granular size of 0.1 μm or less, are evenly present.    Neutrophil: A large number of glycogen granules ranging from 0.05 to 0.2 μm are present over a cell.    Eosinophil: A large number of special granules ranging from 0.5 to 1.0 μm are present.    Basophil: A large number of granules ranging from 0.2 to 1.0 μm are present.
These cells increase or decrease in number in accordance with a disease. Therefore, a disease is diagnosed by detecting the status of each cell on the basis of information about granules or particles in the cell. Therefore, classification of leukocytes is useful in clinical inspection.
In order to classify leukocytes with a flow cytometer, a blood sample including the leukocytes is irradiated by a laser beam originating from a laser light source so that the resultant scattered light, which differs in accordance with the type of a blood cell, is detected.
As disclosed in Japanese Patent No. 3350775 (corresponding to Japanese Patent Publication No. 8-271509A), the basic configuration of the flow cytometer is to classify leukocytes by making a sheath flow from a blood sample containing leukocytes, causing the sheath flow to pass through a tubule; irradiating a laser beam onto the flow of blood sample in the tubule orthogonally thereto; and detecting forward scattered light and orthogonal scattered light.
In U.S. Pat. No. 6,084,670, there is proposed adoption of a Fresnel lens as an optical lens to be used in the flow cytometer.
FIG. 1 shows the structure of a flow system and that of an optical detection system, both being located in the vicinity of a flow cell of the flow cytometer. These structures may be similarly applied to a flow cytometer of the invention.
A sample flow (i.e., a blood sample) 4b flows upward through a flow path formed in a quartz flow cell 4 while being sheathed in a sheath flow 4a. In the sample flow 4b, blood cells flow one after another in a line.
A laser beam 3 is emitted from a laser diode 1 to irradiate the sample flow 4b orthogonally thereto. When the particles flowing in the sample flow are exposed to the laser beam 3, optical scattering arises.
In relation to scattered light, a Fresnel lens 5a for detecting forward scattered light 3a and another Fresnel lens 8 for detecting orthogonal scattered light 3d are provided.
The Fresnel lens 5a is integrally constructed by arranging an inner ring-shaped Fresnel lens and an outer annular Fresnel lens in a concentric manner. A mask 5b is attached to a part of the Fresnel lens 5a facing the flow cell 4 such that the inner ring-shaped Fresnel lens receives forward small scattered light 3b and such that the outer annular Fresnel lens receives forward large scattered light 3c. Here, an angle range in which detection is limited by the mask 5b corresponds to a scattering detection angle range outside the flow cell (described later).
Here, the intensity of the forward small scattered light correlates with the size of the particles (granules or nuclei) that have been exposed to the laser beam. The intensity of the forward large scattered light correlates with the complexity of the exposed particles. The intensity of the orthogonal scattered light correlates with granularity.
Here, the detection angle range of the scattered light at the outside of the flow cell will now be described by reference to FIGS. 4A and 4B.
As shown in FIG. 4A, the detection angle range at the outside of the flow cell is a range defined by representing, as a geometric (linear) angle, an area in which the Fresnel lens 5b detects scattered light while taking as a center a point of scattering at which blood cells in the sample flow 4a flowing through the flow cell 4 are irradiated by a laser beam.
Consequently, a range in which the forward small scattered light 3b is detected corresponds to a range defined between a1 and b1; a range in which the forward large scattered light 3c is detected corresponds to a range defined between c1 and d1; and a range in which the orthogonal scattered light 3d is detected corresponds to a range defined between e1 and f1.
In reality, as shown in FIG. 4B, the scattered light undergoes refraction at a boundary between the inside and outside of the flow cell 4 because of the refractive index of the flow cell 4, the refractive index of the sample flow 4b and the sheath flow 4a flowing through the flow cell 4, and a difference in refractive index between the inside and outside of the flow cell 4. Since the main component of each of the sheath flow 4a and the sample flow 4b is water, the refractive indices thereof are regarded as almost the same.
In view of the above, the scattering detection angle ranges within the flow cell will be considered.
FIG. 5 shows a range defined by the angle through which the laser beam radiated onto the sample flow in the flow cell 4 is scattered. The detection angle range of the forward small scattered light 3b corresponds to the range defined between a2 and b2. The detection angle range of the forward large scattered light 3c corresponds to the range defined between c2 and d2. The detection angle range of the orthogonal scattered light 3d corresponds to the range defined between e2 and f2.
A scattering angle outside of the flow cell and a scattering angle inside of the flow cell differ from each other depending on a refractive index of a liquid in the flow cell 4, a refractive index of the flow cell, and a refractive index of space (air) outside the flow cell. Further, a scattering angle outside of the flow cell and a scattering angle inside the flow cell differ from each other depending on the component and concentration of a reagent for processing blood required to produce a blood sample.
FIG. 2 is a block diagram showing the entire configuration of the flow cytometer.
The laser diode 1 radiates a laser beam 3 in accordance with a signal output from a laser diode controller 2 which has received a control signal from a processor 10. Forward scattered light 3a including forward small scattered light 3b and forward large scattered light 3c is divided by the mask 5b. Both light rays are collected by the Fresnel lens 5a and detected by detectors 6, 7.
Orthogonal scattered light 3d is collected by a Fresnel lens 8 and detected by a detector 9. The intensity of each of the scattered light rays detected by the detectors 6, 7, and 9 is transmitted to the processor 10. The processor 10 plots a distribution on a scattergram based on the detection signals, thereby classifying leukocytes.
The mask 5b is constructed as shown in FIG. 3. Reference numeral 3b′ designates an area which enables passage of the forward small scattered light 3b, and 3c′ designates an area which enables transmission of passage of the forward large scattered light 3c. 
A cross pattern provided at the center of the mask 5b is provided for preventing detection of direct light of the laser beam 3 other than the light rays scattered by blood cells.
As mentioned above, there has been proposed a method of enhancing accuracy of classification of leukocytes, by detecting the forward scattered light which is divided into forward small scattered light and forward large scattered light.
Japanese Patent Publication. No. 8-50089A also discloses the classification of leukocytes by detecting forward small scattered light and forward large scattered light. Specifically, scattered light falling within a region ranging from 1 to 5 degrees with respect to the optical axis of the incident light is detected as forward small scattered light, and scattered light falling within a region ranging from 6 to 20 degrees with respect to the optical axis of the incident light is detected as forward large scattered light. However, reasons why these regions are determined as detection angle ranges are not evident.
In addition to containing lymphocytes, monocytes, neutrophils, eosinophils, and basophils, blood sometimes contains immature leukocytes such as immature granulocytes including myeloblasts or myelocytes, or immature erythrocytes such as erythroblasts, all of which are usually present in bone marrow, but not in peripheral blood. Further, abnormal leukocytes, such as lymphoblasts, deformed lymphocytes, and abnormal lymphocytes, also sometimes emerge. Therefore, there is a necessity of identifying these immature or abnormal blood cells, thereby classifying leukocytes.
As has been known, an angle distribution of scattered light differs according to the size of particles when granules and particles are exposed to a laser beam. The light scattered by large particles substantially appear at a forward areas in a concentrated manner, and the intensity of the scattered light is proportional to the square of the diameter of a particle (according to Fraunhofer's diffraction theory). If particles have small diameters, the scattered light spreads in all directions, and the intensity of scattered light is proportional to the sixth power of the diameter of the corresponding particle (according to the Rayleigh's theory). The light scattered by small particles differs in angle distribution with respect to the oscillatory direction of incident light. As has also been known, if the particles have an irregular surface, a polarization phenomenon due to scattering does not arise.
When a laser beam having a wavelength λ is radiated onto particles which are included in leukocyte cells and have a diameter “d,” a particle parameter can be expressed as:α=πd/λ  (1)given that the particle parameter is defined as α.
The angle distribution of scattered light intensity can be expressed as shown in FIG. 13 according to the oscillatory direction of incident light. It is assumed that linearly-polarized incident light (i.e., a laser beam 3) enters along the Z axis and is scattered by a particle of leukocyte cell placed at the point of origin, in the direction of θ (scattering angle) with respect to the Z axis (here, the scattered light is represented by an arrow 3a). A plane parallel to Y-Z plane is taken as a view plane. The incident light is assumed such that an oscillatory direction of an electric field is linearly polarized in parallel with X-Z plane, and an oscillatory direction of a magnetic field is linearly polarized in parallel with the view plane (i.e., Y-Z plane).
Scattered light intensity is obtained from the light scattered by the particle along oscillatory planes. Here, the intensity of scattered light having a component perpendicular to the view plane (i.e., X-Z plane) is taken as I1, and the intensity of scattered light having a component parallel to the view plane (i.e., Y-X plane) is taken as I2. According to the Mie's scattering theory, the intensity of scattered light can be calculated by the following equation.
                    I        =                              ∫                          ω              ⁢                                                          ⁢              c                                ⁢                                                    F                ⁡                                  (                                      θ                    ,                    ψ                    ,                    α                    ,                    m                                    )                                            K2R2                        ⁢                                                  ⁢            I            ⁢                                                  ⁢            ⅈ            ⁢                          ⅆ              ω                                                          (        2        )            where α=πdp/λ    K=2π/λ    I: intensity of scattered light    II: intensity of incident light (hereinafter called “incident light intensity”)    λ: wavelength    dp: particle size (“p” means particle)    ωc: light focusing solid angle    m: relative refractive index between a medium and a particle    R: distance from a particle to an observation point    θ, ψ: incoming and outgoing directions of light in a spherical coordinate system
As shown in FIGS. 14A and 14B, the thus-computed scattered light intensities I1 and I2 shows distributions on the basis of the scattering angle θ of the particle. Since the scattered light intensity I1 changes according to an angle at an electric field oscillation plane, it is obtained a distribution in the form of a pair of eyeglasses, such as that shown in FIG. 14A. The scattered light intensity I2 is identical at any angle on a magnetic field oscillation plane. Hence, it is obtained a distribution in the form of a circle such as that shown in FIG. 14B.
In a case where the incident light is not linearly polarized, the scattered light intensities I1 and I2 are added, whereby a merged distribution such as that shown in FIG. 14C is obtained. The distribution pattern of merged scattered light intensities changes according to the value of the particle size parameter α of Equation 1. The left side of the coordinate system with respect to the center in FIG. 14C shows a pattern of a forward scattered light component, and upper and lower patterns show patterns of an orthogonal scattered light component. The right side of the coordinate system with respect to the center shows a pattern of backward scattered light components. When the particle size parameter α is 0 or very small, a laterally-symmetric pattern as illustrated is obtained. Further, all these patterns are vertically symmetric.
FIGS. 15A to 15D show changes in particle size parameter α; that is, changes in the sizes of particles in a cell, wherein
FIG. 15A shows a pattern for a particle size parameter α=0.6;
FIG. 15B shows a pattern for a particle size parameter α=1.0;
FIG. 15C shows a pattern for a particle size parameter α=1.5; and
FIG. 15D shows a pattern for a particle size parameter α=3.0.
As is evident from these figures, a backward scattered light component becomes smaller as the particle size parameter α increases; that is, as a particle becomes larger. On the other hand, a forward scattered light component extends in the direction of the incident light axis as a particle becomes larger.
A scattergram employing the intensity of detected forward small scattered light and the intensity of detected forward large scattered light as axes is defined as a scattergram denoted by SC1 shown in FIG. 16. In the scattergram SC1, the respective reference characters represent as follows:                Ne: neutrophil        Ly: lymphocyte        Mo: monocyte        Eo: eosinophil        Ba: basophil        
In order to separate and analyze the monocytes, the basophils, and the lymphocytes in more detail, a scattergram SC2 is prepared based on the forward small scattered light intensity range in the scattergram SC1 where monocytes, basophils, and lymphocytes are distributed and an appropriate orthogonal scattered light intensity range.
In order to separate and analyze the neutrophils and the eosinophils in more detail, a scattergram SC3 is prepared based on the forward small scattered light intensity range in the scattergram SC1 where neutrophils and eosinophils are distributed and an appropriate orthogonal scattered light intensity range.
In the related-art flow cytometer, with respect to the irradiating direction of laser beam 3, the range in which the forward small scattered light 3b is to be detected, the range in which the forward large scattered light 3c is to be detected, and the range in which the orthogonal light 3d is to be detected are determined as shown in Table 1. It is adopted a reagent sodium polyoxyethylene 3 alkyle (C12-C13 mixture) ether sulfate [i.e., a reagent using a chemical formula C12-13—O—(CH2CH2O)3—SO3Na] which serves as a hemolytic agent compound and is a kind of polyoxyethylene anionic active agent is disclosed as EXAMPLE 1 (a hemolytic agent component) in U.S. Pat. No. 5,747,343.
TABLE 1for forward smallfor forward largefor orthogonalscattered lightscattered lightscattered lightdetection angle0.9-5.010-1663-117range outside(degrees)(degrees)(degrees)flow celldetection angle0.7-3.9 8-1269-111range inside(degrees)(degrees)(degrees)flow cell
The “detection angle range outside the flow cell” provided in an upper row of Table 1 is a range defined by representing, as a geometric (linear) angle, an area in which the Fresnel lens 5b detects scattered light while taking as a center a point of scattering at which the blood cells in the sample flow 4b is irradiated by the laser beam 3 (see FIG. 4A).
The “detection angle range inside the flow cell” provided in a lower row of Table 1 is a range in which the Fresnel lens 5b actually detects scattered light while taking as a center a point of scattering at which the blood cells in the sample flow 4b is irradiated by the laser beam 3 (see FIGS. 4B and 5).
Therefore, the scattered light detection angle range outside the flow cell may slightly deviate from the scattered light detection angle range inside the flow cell, depending on a refractive index of the flow cell 4, a refractive index of the interior or exterior of the flow cell 4, and the design dimensions of the flow cell 4.
The scattergram obtained by the configurations shown in Table 1 will be described layer. Here, substantially the same result can be obtained through use of a reagent disclosed as EXAMPLE 2 (a hemolytic agent component) in U.S. Pat. No. 5,747,343, wherein a reagent sodium polyoxyethylene 3 alkyle (C11-C15 mixture) ether sulfate serves as a hemolytic agent compound and is a kind of polyoxyethylene anionic active agent.
However, in the scattergram plotted with the intensity of the forward large scattered light and the intensity of the forward small scattered light are taken as axes, lymphocytes and monocytes are distributed in close proximity to each other. If these distributions are separated from each other, accurate measurement can be performed.
Since the neutrophils and eosinophils are in close proximity to each other on the scattergram, accurate measurement can be performed, so long as the distributions can be separated from each other.
When large immature cells have arisen, the distribution of the neutrophils and a distribution of the large immature cells are close to each other on the scattergram using the intensity of the forward large scattered light and the intensity of the forward small scattered light as axes. Hence, confusion of measurement may arise between these two types of cells.